Elg1, a central player in genome stability

Mutation Research 763 (2015) 267–279

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Review

Elg1, a central player in genome stability Inbal Gazy a, Batia Liefshitz a, Oren Parnas b, Martin Kupiec a,* a b

Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Israel Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 September 2014 Received in revised form 15 November 2014 Accepted 17 November 2014 Available online 24 November 2014

ELG1 is a conserved gene uncovered in a number of genetic screens in yeast aimed at identifying factors important in the maintenance of genome stability. Elg1’s activity prevents gross chromosomal rearrangements, maintains proper telomere length regulation, helps repairing DNA damage created by a number of genotoxins and participates in sister chromatid cohesion. Elg1 is evolutionarily conserved, and its mammalian ortholog (also known as ATAD5) is embryonic lethal when lost in mice, acts as a tumor suppressor in mice and humans, exhibits physical interactions with components of the human Fanconi Anemia pathway and may be responsible for some of the phenotypes associated with neurofibromatosis. In this review, we summarize the information available on Elg1-related activities in yeast and mammals, and present models to explain how the different phenotypes observed in the absence of Elg1 activity are related. ß 2014 Elsevier B.V. All rights reserved.

Keywords: Genome stability DNA replication DNA repair Telomere length regulation Chromatin Sister chromatid cohesion

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Genome stability and the DDR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The RFC-like complexes (RLCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rad24 RLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Ctf18 RLC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Elg1-RLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Elg1 and SUMO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roles of Elg1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Elg1 and DNA replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Elg1 and DNA repair mechanisms . . . . . . . . . . . . . . . . . . . . . . 4.2. Double strand break (DSB) repair . . . . . . . . . . . . . . . 4.2.1. Gross chromosomal rearrangements . . . . . . . . . . . . 4.2.2. DNA damage tolerance (DDT) pathway . . . . . . . . . . 4.2.3. Srs2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.4. Fanconi Anemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.5. Sister chromatid cohesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Telomere length maintenance . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Are all the phenotypes of Delg1 dependent on PCNA modification? Elg1 in mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Genome stability and the DDR * Corresponding author. Tel.: +972 3 640 9031; fax: +972 3 640 9407. E-mail address: [email protected] (M. Kupiec). http://dx.doi.org/10.1016/j.mrrev.2014.11.007 1383-5742/ß 2014 Elsevier B.V. All rights reserved.

A stable genome is a pre-requisite for the survival and proper functioning of all organisms. Genome stability is compromised

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constantly by external insults (such as DNA damaging agents) and may be even threatened by the normal metabolism of the cell. In addition, in each cell division it is necessary to duplicate the genetic material; during DNA replication the double stranded helix must be open to allow its copying, a situation that renders DNA prone to chemical modifications and alterations. Several surveillance and repair mechanisms operate in eukaryotic cells to ensure the stability of the genome, and dire consequences to the cell ensue when they fail to act properly. Indeed, genomic instability is a hallmark of cancer cells. Most human cancer cells show signs of genome instability, ranging from elevated mutation rates, to gross chromosomal rearrangements, including deletions and translocations. The current view is that most spontaneous chromosomal rearrangements and mutations in the genome arise during DNA replication. The activity of the DNA polymerases may be impaired by the presence of secondary structures, bound proteins or DNA lesions; they may also be halted by collisions with other DNAinteracting proteins (such as RNA polymerases, or topoisomerases). These encounters may lead to stalling or even collapse of replication forks, creating single-stranded gaps or double-strand breaks (DSBs). In response, cellular mechanisms are activated that arrest cell cycle progression, induce DNA repair or lesion bypass, and restore replication, in what is commonly called the ‘‘DNA damage checkpoint’’ or the ‘‘DNA damage response’’ (DDR). The checkpoint pathways are activated by delays in DNA replication (replication stress pathway) or by DNA lesions that obstruct replication (DNA damage pathway) [1,2]. This protective cellular response is conserved, which underscores its centrality and importance. Because of this conservation, simple organisms, such as budding yeast, are extremely useful for studying the basic principles of genome stability and maintenance. Not surprisingly, genes that were found to affect genome stability in yeast were later found to be tumor suppressors in human cells. Unless otherwise specified, we will hereafter refer to the vast knowledge on the mechanisms that maintain genome stability accumulated in yeast. PCNA is a homotrimeric ring that encircles the double stranded DNA and plays a central role in DNA replication. It acts as a processivity factor for the replicative DNA polymerases, and serves as a moving platform during DNA replication to which DNA interacting proteins can bind. Many proteins are known to interact with PCNA, including factors involved in DNA replication, DNA repair, chromatin remodeling and other DNA-related activities that are important for cell viability, cell division, and genomic stability. Many of these proteins interact with PCNA via a PCNA-interacting peptide (PIP) or motif [3]. When DNA is damaged, PCNA may be mono-ubiquitinated at lysine 164 by the E2/E3 pair Rad6 and Rad18 [4]. This modification of PCNA activates the DNA Damage Tolerance pathway (DDT, also known as post-replication repair or PRR). Mono-ubiquitination of lysine 164 allows the binding of special DNA polymerases able to replicate damaged DNA molecules, at the expense of accuracy. These translesion synthesis polymerases participate in an error-prone bypass mechanism that results in the creation of mutations. Alternatively, PCNA can be further poly-ubiquitinated on the same lysine residue by an alternative DDT mechanism that also requires an E2 heterodimer composed of the Ubc13 and Mms2 proteins and the E3 protein Rad5 [5]. This poly-ubiquitination coordinates an error-free repair mechanism, whose details are still unclear [6]. Interestingly, the same residue of PCNA (lysine 164) can be modified by the ubiquitin-like molecule SUMO. This modification takes place during S-phase or after high doses of DNA damage and requires the SUMO-specific E2 Ubc9 and the SUMO ligase Siz1 [4]; short SUMO chains are sometimes seen, but their significance is still unclear [7]. An additional residue, lysine 127, can also be SUMOylated, but not ubiquitinated. In contrast to mutations in lysine 164, those in lysine 127 do not lead to DNA damage sensitive

phenotypes [4]. SUMOylation of PCNA strongly affects the choice of pathway used for processing the lesions. SUMOylation seems to prevent homologous recombination, favoring ubiquitin-dependent lesion bypass [8,9]. Thus, PRR mutants, such as Drad5, Dmms2 or Dubc13 exhibit a high level of spontaneous mutations, caused by channeling of lesions to the trans-lesion synthesis pathway. This mutagenesis can be reduced by preventing PCNA SUMOylation, for example, by deleting the E3 SUMO ligase SIZ1. In these PRR defective backgrounds, the lesions are channeled to error free recombinational pathways and mutation levels are reduced [10,11]. 2. The RFC-like complexes (RLCs) Replication factor C (RFC) is a 5-subunit complex in charge of loading PCNA onto DNA to allow its replication. RFC is composed of a large subunit, Rfc1, and four small subunits (Rfc2–5). In vitro, RFC is capable of both loading and unloading PCNA onto DNA, although it is believed that its main function is to load the clamp, a function required for accurate replication and repair of the genome [12]. Mutations in the yeast ELG1 gene lead to a variety of genomic instability phenotypes, including, among others, hyper-recombination, hyper-transposition, chromosome loss, gross chromosomal rearrangements, elongated telomeres and increased telomeric silencing [10–24]. The Elg1 protein resembles Rfc1, the large subunit of replication factor C, and forms an RFC-like complex (RLC) together with the four small RFC proteins, Rfc2–5 [11–13] (Fig. 1). Elg1 RLC, as will be discussed below, can unload PCNA from DNA during replication. Two additional RLCs can be detected in most organisms: both are composed of the small RFC subunits, but have the large Rfc1subunit replaced by the proteins Rad24 or Ctf18 (Fig. 1). The canonical RFC complex is essential, as it is required for polymerase loading during DNA replication. In contrast, none of the alternative RLCs is essential, alone or in combinations. The triple Delg1 Drad24 Dctf18 mutant is viable, although it grows very poorly and shows extremely high levels of genome instability and sensitivity to DNA damaging agents [12]. 2.1. Rad24 RLC The first RLC to be found was Rad24 RLC [13]. Interestingly, this evolutionarily conserved complex loads an alternative clamp composed of three different proteins, Rad17/Mec3/Ddc1, that forms a PCNA-like heterotrimer (the clamp in humans is called the 9-1-1 complex) [14]. The Rad24-RLC loads the alternative clamp on partial duplex DNA and activates the DNA damage checkpoint activation and repair. The loading mechanism of Rad24 RLC/9-1-1 is similar to that of RFC/PCNA, although the DNA substrate requirements are different [15,16]. In vitro experiments demonstrated that only after proper loading of the 9-1-1 clamp by Rad24, Mec1 (the yeast ortholog of ATR) binds and phosphorylates the clamp subunits, as well as the Rad53 protein (the yeast ortholog of Chk2), thus eliciting the DNA damage response [15]. Thus, the Rad24 RLC seems to be responsible for the first step in checkpoint activation [17]. No actual DNA damage is required: in fact, it is enough to load the 9-1-1 complex and Mec1-Ddc2 to the chromatin to obtain a full DDR in the absence of DNA damage [18]. In addition, loading of the 9-1-1 clamp seems to be necessary for proper induction of the error-free DDT pathway [19]. 2.2. Ctf18 RLC In the second RLC, the four small Rfc subunits (Rfc2–5) interact with Ctf18 [20,21]. Mutations in CTF18 were found in two independent screens for genes important for prevention of chromosome loss [22,23], and later, in a specific screen for mutants affecting origin of replication firing [24]. Dctf18 mutants are

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Rfc2-5

269

Dcc1 Ctf8

Clamp loader Rfc1

Elg1

Rad24

Ctf18

Clamp PCNA

Function

DNA synthesis. Main loader and unloader of PCNA.

PCNA Genome stability. Alternative unloader of PCNA.

9-1-1 DNA damage checkpoint.

PCNA

Sister chromatid cohesion.

Fig. 1. RFC and RFC-like (RLC) complexes. The three conserved RLCs share with RFC the small subunits (Rfc2–5) (in gray), and replace the large subunit by either Elg1, Rad24 or Ctf18, respectively. The Ctf18 RLC also contains two additional subunits, Dcc1 and Ctf8. While the canonical RFC as well as Elg1 RLC and Ctf18 RLC interact with the DNA clamp PCNA, Rad24 RLC loads an alternative clamp named 9-1-1.

sensitive to drugs that cause tubulin depolymerization, such as benomyl and nocodazol [22,23]. They exhibit elevated levels of recombination [25] and share genetic interactions with genes involved in DNA replication [26]. Interestingly, the Ctf18 RLC complex contains two additional subunits, Dcc1 and Ctf8, which are unique to this alternative RLC complex [21]. The seven subunit complex is conserved also in humans [27] and was shown to interact with PCNA both in humans and in yeast [28]. However, the nature of this interaction is not yet clear. In Dctf18 mutants there is a reduction in the amount of chromatin-bound PCNA, which may imply that it loads PCNA on the DNA [29]. However, in vitro experiments with Ctf18-RLC show both loading [30] and unloading activity [28]. Human Ctf18 RLC was also found to be important for replication fork velocity [31] and this effect of Ctf18 seems to be linked to its major role in sister chromatid cohesion (SCC) (see below). In yeast, the Ctf18 RLC (but not the Elg1-nor Rad24 RLCs) were found to be essential for activation of the DNA replication checkpoint [32]. Similar conclusions were reached using a proteomic approach [33]. Although Ctf18 has a clear role in SCC, it is important to mention a recent report claiming that Ctf18 has additional roles in genome stability that are independent of its role in SCC [34]. 2.3. Elg1-RLC ELG1 shows sequence homology to RFC1 and is always isolated as part of a 5-protein complex together with the small RFC subunits (Rfc2–5) [17,35–37]. ELG1 was found in several genomewide screens in yeast [35,36,38–41]. Delg1 mutants show elevated rates of homologous recombination [35,42], chromosome loss [35] and gross chromosomal rearrangements [43]. They exhibit elongated telomeres [41,44] and increased levels of Ty transposition [40]. Thus, mutations in ELG1 greatly increase genomic instability. Consistent with this idea Delg1 mutants show elevated levels of spontaneous Ddc2 foci [45] and Rad52 foci [46] that point to the existence of spontaneous DNA damage, probably created during DNA replication of cycling cells. Homologous recombination (HR) is highly increased in Delg1 mutants: all types of recombination tested (direct repeat recombination, gene conversion, allelic and ectopic, sister chromatid recombination, etc.) appear to be elevated in vegetative, but not meiotic, cells in the absence of Elg1 [35]. The general effect of Delg1 mutants on HR suggests that Elg1 is not involved in suppressing a specific HR branch but has a more general role in the maintenance of genomic stability. Interestingly, in the presence of exogenous DNA damaging drugs [methylmethane sulfonate (MMS) or phleomycin] elg1 mutants exhibit a

lower induction of HR than wild type cells [42]; thus, the Elg1 protein may, under certain circumstances, be required to promote HR. 3. Elg1 and SUMO SUMO is a small protein whose mechanism of activation and ligation to proteins is similar to those of ubiquitin [47]. Yeast cells express a single E1 enzyme (Uba2/Aos1), a unique E2 enzyme (Ubc9) and three known E3 enzymes (Siz1, Siz2 and Mms21). SUMO has been shown to affect a variety of processes in all eukaryotes, including sister chromatin cohesion, transcription and DNA repair [47,48]. Although ubiquitin and SUMO have usually distinct cellular functions, in some cases they interact: SUMOylation of particular proteins serve as a signal for their ubiquitination by the Slx5/Slx8 heterodimer and eventually for their degradation [49]. This complex (encoded by a homodimer of RNF4 proteins in humans [50]) forms an E3 ubiquitin ligase that selectively ubiquitinates SUMOylated and poly-SUMOylated proteins [51,52]. The interactions of Slx5/Slx8 with the SUMOylated substrates are mediated by SUMO-interacting (SIM) motifs in Slx5 [49]. These motifs are composed of small patches of hydrophobic amino acids followed by negatively charged amino acids. In vitro studies showed that following the interaction of Slx5 with the SUMOylated substrate, the E3 ubiquitin ligase activity of Slx8 is necessary to ubiquitinate the SUMO chains [51,52]. The Slx5/8 complex was thus defined as a SUMO-Targeted Ubiquitin Ligase (STUbL). Strains deleted for the SLX5 or SLX8 genes show an accumulation of high molecular weight SUMOylated proteins [49]. This accumulation is also shared by proteasome mutants; the epistatic relation between these mutants raises the possibility that the Slx5/8 complex belongs to a pathway that sends proteins to the proteasome for degradation. It should be noted, however, that only very few substrates of the E3 ubiquitin ligase complex Slx5/Slx8 have been identified to date, and the ubiquitination may serve signaling purposes other than degradation. Deletion of SLX5 or SLX8 in yeast causes high levels of homologous recombination [53], elevated levels of spontaneous gross chromosomal rearrangements [54] and other phenotypes of genomic instability. Slx5/8 was shown to be recruited to DNA damage sites [55], suggesting a role in the response to DNA damage. An unbiased genome-wide yeast two hybrid screen uncovered physical interactions between Elg1’s N-terminal domain and SUMO, as well as with the SUMO E2 Ubc9 and with Slx5 [56]. Further analysis uncovered that the interactions between Elg1 and Slx5 required the presence of intact SIM motifs in both

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Slx5 and Elg1, and were abolished in a strain lacking the E3 SUMO enzyme Siz2. The interaction was also abolished in a strain in which all the lysines of SUMO were replaced by arginine residues, so that it is able to carry out mono but not poly-SUMOylation. Taken together, these results suggest a model in which the interaction between Elg1 and Slx5/8 is mediated by a still-unidentified poly-SUMOylated protein. Interestingly, the interaction was not affected in strains unable to modify their PCNA, suggesting that the role of Elg1 in its interactions with the STUbL are distinct from those needed to interact with PCNA (see below). It has been suggested [57] that SUMO may act as a general ‘‘glue’’ to hold together different components of the same pathway located at the same location and time, thus coordinating the activity of enzymes. Once the activity is completed (e.g. DNA repair or replication re-initiation in this case), SUMO needs to be removed from all these proteins, and STUbL activity (ubiquitin marking of SUMOylated proteins) may be required for their degradation. 4. Roles of Elg1 Below, we dissect the various processes in which Elg1-RLC participates. Clearly, the division is artificial, as they are all interconnected and dependent on each other.

Delg1 cells are dependent on the homologous recombination pathway for survival and exhibit synthetic lethality with mutants of the RAD52 group, or mutations in nucleases such as Mms4 and Mus81 [36,37]. Moreover, synthetic genetic interactions could also be seen between Delg1 and late Okazaki fragment processing mutants such as Drad27 and dna2 [37,65] and physical interactions of Elg1 with Rad27 could also be detected [37]. However, it is important to point out that the Elg1 RLC cannot be the only mechanism able to unload PCNA, as mutants deleted for ELG1 show only slight growth defects [36,37] and, despite interactions with Okazaki fragment maturation components, Okazaki fragments appear normal in Delg1 mutants [63]. Hence, the consequences of PCNA’s accumulation on chromatin seem to emerge only after passage of the fork, possibly through the recruitment of interaction partners that may initiate inappropriate repair or recombinationrelated events. An alternative possibility is that Elg1 may be responsible for the unloading of PCNA at specific genomic regions, such as cohesion sites [66,67], with the canonical RFC, or Ctf18 RLC unloading PCNA from other genomic regions. PCNA retention on chromatin in the absence of Elg1 activity may cause genome instability through altered recruitment of PCNA-interacting factors

4.1. Elg1 and DNA replication Genetic screens in yeast have shown that Delg1 mutants have synthetic growth defects when combined with genes involved in sister chromatid cohesion (SCC), checkpoint response, replication fork restart and homologous recombination [36,58–60]. These genetic interactions suggest that in the absence of Elg1, the cells may experience problems during DNA replication that may require their repair, as well as replication restart. Elg1 was shown to play a role in the intra-S checkpoint that prevents firing of late replication origins when the DNA is damaged; the phenotype of the Delg1 mutant, however, was milder, compared to that of a Dctf18 mutant [32], suggesting that the Ctf18-RLC may be playing a more important role in this process. In Delg1 mutants there is a slight delay in S phase progression [36,37]; however, this phenotype is relatively minor compared to mutations in known replication proteins. Moreover, Okazaki fragments are completed normally and replication proceeds without activating a damage signal [61]. Thus, it is unlikely that Elg1 plays a central role as clamp loader during DNA replication; that role seems to be entirely attributed to the canonical RFC complex. Moreover, no loading activity could be detected in vitro using purified Elg1 RLC components [28]. Parnas et al. [62] showed that Elg1 is preferentially associated with PCNA, and particularly with SUMOylated PCNA, via interactions between a PIP and 3 SIM motifs located at the N-terminus of Elg1. Moreover, deletion of ELG1 resulted in the accumulation of SUMOylated PCNA on the chromatin, suggesting a role for Elg1 in either PCNA unloading or de-SUMOylation [62]. Proteomic and biochemical work by the Donaldson lab support this model: using a degron system in which Elg1 can be swiftly eliminated from the cells, they showed that during replication, PCNA accumulates on the chromatin in the absence of Elg1. The accumulated PCNA could be removed from chromatin in vivo by switching on Elg1 expression. In addition, treating chromatin with purified Elg1 RLC in vitro resulted in PCNA unloading [63]. Taken together, these results suggest a role for the Elg1-RLC in PCNA unloading, possibly during DNA replication, but more likely as a response to fork stalling or collapse. As most forms of DNA repair and damage bypass require DNA synthesis, PCNA participates in repair, and needs to be loaded and unloaded there too [64]. Indeed, Delg1 mutants exhibit defects in mechanisms that can re-initiate replication after stalling. As a consequence,

Fig. 2. Different potential functions of Elg1. (A) During DNA replication, Elg1 unloads PCNA molecules, particularly those that are SUMOylated. PCNA SUMOylation may serve as a signal for Okazaki fragment processing. Cohesin rings are schematically depicted before, and after replication fork passage. For simplicity, other proteins present at the fork are omitted. (B) At telomeres, Elg1 may coordinate telomere elongation. Telomerase elongates the leading strand, and the lagging strand is coordinately completed. The CST (Cdc13-Stn1-Ten1) complex helps loading polymerase alpha, which interacts with accessory telomerase subunits. (C) During replication, nucleosomes in front of the advanced fork are displaced, and their histone proteins used, together with new histones, behind the moving fork. New histones are marked by acetylation of lysine 56 at histone H3. This modification is removed, in coordination with Okazaki fragment maturation, by the Hst3–Hst4 sirtuins.

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including cohesion factors, histone chaperones, and DNA repair proteins (see below [3]). PCNA unloading may also be necessary to exchange DNA polymerases: during DNA replication origin firing is carried out by primase-Pola, and later changed to the replicative polymerases, Pold and Pole. This switch may be affected by lack of Elg1 activity (Fig. 2). 4.2. Elg1 and DNA repair mechanisms 4.2.1. Double strand break (DSB) repair One of the most dangerous types of DNA lesion is a doublestranded DNA break. If left unrepaired, part of the genome is loss during cell division; if improperly repaired, it may lead to gross chromosomal rearrangements and/or cell death [68,69]. DSBs are repaired by two competing mechanisms, non-homologous end joining (NHEJ), which ligates broken DNA ends irrespectively of sequence, and homologous recombination (HR), which uses genomic sequences similar to the broken ends as templates to synthesize DNA across the break, thus restoring the broken information. This pathway requires a number of proteins, including Rad52 and Rad51 [68,70]. Ogiwara and colleagues [42] reported that Elg1 is recruited to broken DNA ends, where it plays a role in repair. They observed a reduced induction of recombination in Delg1 mutant cells exposed to DNA damaging agents such as MMS and phleomycin compared to wt cells. In a system carrying a single, inducible DSBs, they observed recruitment of Elg1 to the broken ends, which was independent of the presence of the Rad52 protein. No defect was seen in PCNA recruitment in Delg1 mutants (needed for DNA synthesis to complete the repair), but late steps in HR were defective. Additionally, deleting ELG1 had no effect on the efficiency of NHEJ. Taken together, these results support a direct role of Elg1 in DSB repair by HR; however, spontaneous recombination levels are increased in Delg1 mutants [35,42]. Thus, either the different behavior of the mutant is due to the fact that most spontaneous lesions are not DSBs, or there is a fundamental difference between recombination that occurs during regular cell proliferation, and that induced by DNA damaging agents [71]. In the first scenario, Elg1 plays a positive role in DSB repair, but a negative one in HR initiated by other lesions (such as ssDNA nicks or gaps); in the second scenario, Elg1 participates in a damageinduced HR pathway. As Ogiwara et al. [38] saw no changes in the levels of PCNA recruited to the DSB, at this stage it is hard to reconcile the possible camp loading/unloading activity of Elg1 with either scenario. Notably, MMS caused hyper-induction of gross chromosomal rearrangements in Delg1, rather than reduced rates [see below]. 4.2.2. Gross chromosomal rearrangements Various types of gross chromosomal rearrangements (GCRs), including translocations, deletions, de novo telomere additions, and chromosome fusions, are observed in many cancers. Several genes important to prevent GCRs have been identified. These include genes that encode components of the DDR, chromatin remodelers, and genes that participate in DNA repair and telomere maintenance [72,73]. Mutations in ELG1 were identified in genome-wide screens looking for mutants with increased levels of GCR [43]. Further analysis showed that DNA damage (such as MMS treatment) caused an increase in GCRs in Delg1 mutants well above that seen in wt cells [38]. Double-mutant analysis showed synergistic interactions of Delg1 with DDR mutants such as mec1 (human ATR), rad53 (CHK2), chk1 (CHK1), sgs1 (WRN) and rad24 (RAD17). This implies that part of the GCRs in Delg1 strains are usually prevented by the DDR pathway. Dpb11 and Dun1 participate in a DNA replication-dependent branch of GCR prevention, which is different from that of most DDR genes

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[74,75]. Interestingly, deletion of ELG1 reduced the level of GCR observed in dpb11-1(TopBP1) and Ddun1 mutants to that of the single Delg1 mutant. These results thus imply that Elg1 activity is necessary for GCR formation in these mutant strains. Dpb11 is a protein essential for DNA replication, which is also essential for eliciting the DDR. Recent results showed that Dpb11 coordinates checkpoint signal transduction both temporally and spatially, by interacting with Mec1. This kinase phosphorylates (and activates) the Rad9 checkpoint mediator only when it is part of a complex carrying Dpb11, Mec1 and Rad9. To allow the formation of this complex, Rad9 must be phosphorylated by the cyclin-dependent kinase (CDK). Checkpoint signaling via Dpb11, therefore, does not efficiently occur during G1 phase when CDK is inactive [76]. Dun1 is a kinase that plays various roles during S-phase; among them, it controls the levels of dNTPs in the cell [77]. Interestingly, an additional deletion of RAD24 in Delg1 dpb11-1 and Delg1 Ddun1 mutants increased the GCR rates to levels even higher than those observed in the dpb11-1 and Ddun1 mutant strains. Taken together, these results place Elg1 in the replicative pathway of GCR prevention, but also imply that Elg1, under certain circumstances, may participate in GCR creation [38]. 4.2.3. DNA damage tolerance (DDT) pathway As explained above, mono-ubiquitination of PCNA at its lysine 164 residue by the Rad6/Rad18 complex activates the DNA damage tolerance pathway, which allows the damage bypass by two different mechanisms: (1) mono-ubiquitinated PCNA recruits errorprone trans-lesion synthesis polymerases able to utilize DNA substrates possessing a variety of lesion types. (2) Further polyubiquitination by the Rad5, Ubc13 and Mms2 proteins facilitates error free DDT by a still not completely defined pathway [78]. When exposed to DNA damaging agents, Delg1 mutants display a mild sensitivity; surprisingly, deletion of ELG1 suppresses the (higher) sensitivity to DNA damaging agents of Drad5. Rad5 is a large protein that contains a helicase domain and an E3 ubiquitin ligase domain [79]. Delg1 suppressed a rad5 E3 ubiquitin ligase mutant (rad5-E3), as well as Dubc13 and Dmms2 mutations (which affect the error-free DDT pathway), but showed an additive effect with a rad5 mutant defective for its helicase activity (rad5-h) (which affects the error-prone pathway) [62]. Consistently, mutations in genes encoding trans-lesion polymerases exhibited additive effects with deletion of ELG1. Thus, Elg1 activity becomes toxic in the absence of the error-free DDT pathway. This toxicity depended on PCNA SUMOylation, suggesting that PCNA unloading under these conditions becomes toxic for the cell [62]. Recent work from the Ulrich laboratory has shown that Rad18, the E3 ubiquitin ligase that mono-ubiquitinates PCNA, shows enhanced activity when PCNA is SUMOylated [80,81]. These results imply that the activity of Elg1 as an unloader of SUMOylated PCNA may act as a modulator of the DDT pathways, by removing potential substrates. Biochemical experiments are on their way to test this model. 4.2.4. Srs2 Several proteins have been found to interact particularly with SUMOylated PCNA in yeast; among them are Srs2 and Elg1 [9,82,83]. Srs2 is a DNA helicase with homologies to the bacterial helicases Rep, PcrA and UvrD [84]. It is able to unwind DNA substrates containing forks, flaps, D-loops, 50 single stranded DNA overhangs as well as blunt-end double stranded DNA substrates. In addition, Srs2 also has a translocase activity, and is able to displace proteins from ssDNA [85]. It has been shown that in vitro, Srs2 is able to displace the Rad51 strand-exchange protein from ssDNA [86,87], and it is thus many times referred to as an ‘‘anti-recombinase’’. However, depending on the assay used, Srs2 has been shown to promote [88] or prevent [89] homologous recombination. SUMOylated PCNA recruits Srs2 to replication

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forks, where the helicase appears to prevent unscheduled recombination events [8,9]. Similarly to ELG1, mutations in SRS2 suppress the sensitivity to DNA damaging agents of mutants of the error-free DDT pathway [62]. Both proteins are recruited to SUMOylated PCNA by a similar mechanism involving PCNA (PIP) and SUMO-interacting (SIM) motifs [9,82]. Moreover, deletion of ELG1 leads to accumulation of Srs2 at the chromatin fraction, suggesting that Elg1 may play a role in Srs2 unloading, perhaps together with SUMOylated PCNA [62]. Remarkably, however, double deletion of the ELG1 and SRS2 genes results in a synthetic fitness reduction in haploid cells [62] and in complete lethality in diploids [90]. These results imply that at least one of the two proteins must be active to allow cell viability. As both proteins are active during DNA replication, it is likely that they provide alternative mechanisms to deal with specific DNA lesions or replication/repair intermediates, although alternative explanations exist. As described, Srs2 can remove the Rad51 protein from ssDNA, thus preventing potentially hazardous recombination intermediates [80,81]. Recently, a second Srs2-dependent mechanism was described by which SUMO-PCNA and Srs2 block the synthesisdependent extension of recombination intermediates [91]. This new Srs2 activity requires the SIM motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to SUMOylated PCNA somehow dissociates replicative and nonreplicative DNA polymerases (such as Pold and Polh) from the DNA. It is not yet clear which of the two Srs2-mediated activities becomes essential in the absence of Elg1 activity. To better understand the nature of the interaction between Srs2 and Elg1, a genetic screen was performed to find genes that, when overexpressed, are able to rescue the synthetic lethality (SL) between Delg1 and Dsrs2 mutations. This screen uncovered many genes, including 9-1-1 components, the Mph1 helicase (the yeast homolog of FANCM, a human gene that, when mutated, leads to Fanconi Anemia), and the Eco1/Ctf7 sister chromatid cohesion factor [90]. 4.2.5. Fanconi Anemia Fanconi Anemia (FA) is a genomic instability syndrome characterized by bone marrow failure, developmental abnormalities, and increased incidence of cancers [92]. Its surprisingly wide range of clinical findings can be explained by the fact that FA is a chromosomal instability disorder, and cells from FA patients accumulate DNA damage at an increased rate. The most striking cellular hallmark of FA is hypersensitivity to a class of DNA damaging agents that create DNA interstrand crosslinks (ICLs), such as mitomycin C (MMC) or diepoxybutane (DEB) [93]. ICLs are very toxic lesions that prevent DNA unwinding, thereby blocking both DNA replication and transcription. FA is a genetically heterogeneous disease, caused by mutations in at least 15 distinct genes (total number estimated to be around 30). All genes products are believed to function in a common DNA repair signaling pathway which closely cooperates with other DNA repair proteins for resolving DNA ICLs during replication. A central event in the pathway is the mono-ubiquitination of FANCD2 and FANCI upon DNA damage, which is mediated by a group of upstream FA proteins that are assembled into a large nuclear E3 ubiquitin ligase complex, termed the ‘‘FA core complex’’. The mono-ubiquitinated FANCD2/FANCI heterodimer was shown to play multiple roles in the pathway, and to functionally interact with downstream FA proteins such as FANCD1 (or BRCA2), FANCN, and FANCJ, and their associated protein, BRCA1. The activated FA pathway must be inactivated for completion and recycling of the functional pathway, and this event is regulated by the USP1/UAF1 deubiquitinating enzyme complex, which deubiquitinates FANCD2 and FANCI. Disruption of this step leads to elevated levels of FANCD2/

FANCI ubiquitination and DNA repair defects, suggesting a failure in the completion of the FA pathway [94]. Human ELG1 (also known as ATAD5) was found to be associated with USP1/UAF1. In addition to its role in the regulation of PCNA, it may play a role in the regulation of FANCD2/I [95,96]. In yeast the FA pathway is only partially conserved, and includes MPH1 (ortholog of FANCM), CHL1 (FANCJ), MHF1 (MHF1), MHF2 (MHF2) and ELG1 (ELG1) [97–99]. Elg1 physically contacts the Mhf1/Mhf2 histone-like complex and genetically interacts with MPH1 and CHL1 genes. In a study that analyzed the sensitivity of double, triple, quadruple and quintuple mutants to the DNA damaging agent MMS and to hydroxyurea (HU) it was found that genetic interactions show a hierarchy: Chl1 and Elg1 play major roles in the survival to these genotoxins and exhibit synthetic fitness reduction. Mph1 plays a lesser role, and the effect of the Mhf1/2 complex is seen only in the absence of Elg1 on HU-containing medium. Thus, an intricate network of interactions rather than a single, linear repair pathway seems to exist in yeast [97]. 4.3. Sister chromatid cohesion Sister chromatids are held close to each other with the help of a complex called cohesin. The pairing of the chromatids is critical for proper segregation to different daughter cells, and therefore essential for life. Cohesin is composed of four subunits: Smc1 and Smc3 form a huge V-shaped heterodimer [100], which is closed by Mcd1/Scc1, thus forming a ring. The fourth subunit, Scc3, although also essential for chromosome segregation and cell viability, interacts only with Scc1in the outer part of the ring and its contribution to the structure or the function of the cohesin complex is not clear yet. Cohesin complexes are loaded on the DNA during late G1 by the Scc2/Scc4 complex [29] at specific sites along the chromosomes, although actual cohesion is only established later during S phase. During mitosis the kinetochores are bound to the microtubules, establishing bipolar attachments to the spindle apparatus. Cohesin complexes oppose the tension that is implemented by the microtubules until all the chromosomes are bound to the microtubules properly and are arranged in the metaphase plate. Only then, a tightly regulated cascade is activated to trigger the cleavage of Scc1, enabling the separation of sister chromosomes to opposite poles of the nucleus. Defects in cohesin loading, establishment, maintenance or cleavage lead to chromosome loss and cell death. In the last few years it was found that cohesin plays, in addition to chromosome segregation, other important functions in the control of gene expression, higher-order chromosome structure and DNA damage repair (reviewed in [101]). In response to DSBs cohesin is also recruited to chromatin around double strand break sites; this recruitment is important for DSB repair by homologous recombination [102,103], although it is also important to remove it to allow free access to the sister chromatid during repair [104]. A clear linkage exists between DNA replication and proper sister chromatid cohesion. First, Scc2/Scc4 is an essential heterodimer that loads the cohesin complex. Scc2/Scc4 is active only during a short time window in the cell cycle, in late G1 [29] immediately before the cells enter S phase. In some cell types, like Xenopus eggs, the recruitment of Scc2/Scc4 to the chromatin depends on the pre-replication complex [105]. In addition, when cohesin subunits are expressed after DNA replication, they are successfully loaded onto the chromatin but fail to establish sister chromatid cohesion [100]. Second, the Eco1/Ctf7 acetyl transferase is required for cohesion establishment solely during S phase. Eco1 is physically coupled to PCNA [106–108]. Eco1 acetylates Smc3 on conserved lysine residues [109,110]. The acetylation is dependent on the activity of the Scc2/Scc4 complex, suggesting that proper

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loading of cohesin is a pre-requisite for the acetylation [111]. In a reciprocal fashion, Smc3 acetylation is important for replication fork processivity and movement [31]. The acetylation and movement were also found to be dependent on Ctf18-RFC. These data raise the possibility that the RLCs help the replication fork to bypass the loaded cohesin complex [112]. Acetylation of cohesin by Eco1 was also found to be important for the localization of the complex in response to DSBs [113]. Two other proteins that are involved in sister chromatid cohesion are Wpl1/Rad61 and Pds5, which form a complex. In the absence of these proteins the SCC defects observed in eco1 or Dctf18 mutants are suppressed, both in humans [112] and in yeast [114,115]. Thus a model was proposed in which these proteins counteract pro-cohesion activities. Ctf4 is another factor that was found in the same screen with Ctf18 and Eco1/Ctf7 [23] and has an important effect on sister chromatid cohesion [20,116]. Ctf4, together with Ctf18, acts at the replication fork [29], interacts with polymerase a [26] and recruits it to the replication fork [117]. Ctf4 may be responsible for the coordination between different parts of the replication fork [118,119]. Hence, Ctf4 is an important link in the chain that holds these two major cellular processes – sister chromatid cohesion and DNA replication. Dctf4 and Delg1 mutant cells show partially overlapping phenotypes. Both mutants have high levels of recombination, chromosome loss, as well as synthetic growth defects with Drad52, mec1 (the yeast ortholog of ATR) [120,121] and other genes that are involved in DNA metabolism. In addition, they display a striking synthetic lethality: double mutant spores Delg1 Dctf4 germinate, but fail to undergo cell divisions [35,36]. A genetic screen for high copy number suppressors identified the cohesin subunit Mcd1/Scc1 and its loader Scc2 as suppressors of the synthetic lethality between Delg1 and Dctf4, pointing to the fact that the synergistic genetic interaction is due to enhanced defects in sister chromatid cohesion. Consistent with this possibility Delg1 mutants exhibit defects in cohesion and are synthetic lethal with hypomorphic alleles of cohesin subunits (but not with hypomorphic alleles of PDS5) [67]). The role of Ctf18 in SCC has been extensively characterized [20,21]. Microscope-based and biochemical studies indicated that the Elg1-RLC and the Ctf18RLC cooperate in a single pathway for SCC, whereas the Rad24-RLC does not seem to play any role in this process [67]. As explained above, the Eco1 acetylase is essential for cohesion establishment and for sister chromatid cohesion in response to DNA damage. Interestingly, Eco1 mutants unable to interact with PCNA are defective in cohesion and are inviable [106]. Mutations in the lysines 127 or 164 of PCNA, which prevent PCNA SUMOylation, or deletion of ELG1 [66], partially restore the temperature sensitivity of eco1 mutants, suggesting a common SUMO-related suppression mechanism [106]. Consistently, Elg1 overexpression exacerbates its conditional growth defect [66]. In summary, the genetic interactions with cohesin subunits, with the PCNA-coupled Eco1 acetyltransferase and with the forkassociated Ctf4 protein suggest a model in which the Elg1 RLC and Ctf4 mediate the interaction between the DNA replication machinery and the loading/unloading of cohesin (Fig. 2A). Cohesin is loaded onto DNA at the end of G1, but only gets activated upon passage of the replication fork. Thus, during DNA replication, it is expected that the progressing fork would encounter cohesin molecules on its path. A possible model of action of the RLCs and of Ctf4 would be that passage through a cohesin loop may require momentary unloading, then re-loading, of PCNA. This would explain the physical and genetic interactions of these proteins with DNA replication components, as well as with cohesin and its associated proteins. PCNA SUMOylation may thus act as a local signal to coordinate unloading by the Elg1-RLC, and the Ctf18-RLC may participate in re-loading. PCNA SUMOylation has been also

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shown to down-regulate Eco1 acetylation activity [106], either as a signal, or because PCNA unloading following SUMOylation is a prerequisite for cohesin activation. Indeed, Eco1 activity has been linked to Okazaki fragment maturation [122]. In the absence of Elg1 increased levels of SUMOylated PCNA on chromatin would prevent proper Smc3 acetylation by Eco1, resulting in defective establishment of cohesion. Consistently with this two-RLC model of cohesin bypass, Terret and co-workers [31] found that in human cells the Ctf18-RLC controls the speed of replication, and that fork progression was slow when Smc3 was not acetylated by Eco1. Their results suggest a role for the RLCs in allowing replication through already deposited cohesin subunits. Loss of this regulatory mechanism leads to the spontaneous accrual of DNA damage [31]. Interestingly, it has been reported that Chl1, the yeast ortholog of the Fanconi Anemia FANCJ protein, promotes Scc2 loading unto DNA and that both Scc2 and cohesin deposition to chromatin are defective in chl1 mutant cells [123]. This defect could thus explain the synthetic phenotypes observed in Dchl1 Delg1 double mutants [97]. As explained above, the Wpl1/Pds5 complex acts as an antiestablishment factor preventing sister-chromatid cohesion until counteracted in S-phase by the acetyl-transferase activity of Eco1 [124]. Therefore, deletion of WPL1 or PDS5, as deletion of ELG1, suppress the temperature-sensitivity of a conditional eco1-1 mutant [125]. Recent work has shown that Pds5 maintains cohesion, at least in part, by antagonizing the polySUMO-dependent degradation of cohesin [126]. In pds5 mutants, Scc1 is extensively SUMOylated, eliciting an Slx5/Slx8-dependent ubiquitination that sends cohesin to the proteasome for degradation [126]. Detailed genetic analysis has shown that Elg1 participates in a single pathway of antiestablishment, together with Wpl1 and Pds5 [115]. The genetic data implies that Elg1 plays an important role in preventing Smc3 acetylation by the Eco1 acetyltransferase. This result is consistent with a requirement for PCNA SUMOylation in order to allow Eco1 activity, and with the notion that SUMO may serve as a local signal that moves with the replicating fork, allowing the association of Okazaki fragment maturation and ligation, cohesion establishment and proper chromatin packaging. It is interesting to note that the same study [115] also identified the Ctf18-RLC and Chl1 (the yeast ortholog of FANCJ) as pro-establishment factors. Most interestingly, deletion of WPL1 suppressed the synthetic lethality observed between eco1-1 and Dctf18, indicating that the lethality is a result of cohesion defects and not severe DNA replication defects, and suggesting that Eco1 and the Ctf18 RLC work in two separate pathways to establish cohesion [115]. 4.4. Telomere length maintenance Telomeres are nucleoprotein structures that cap the ends of linear chromosomes. Telomeric DNA is comprised of repetitive, noncoding sequences that serve as template for elongation by telomerase, a reverse transcriptase that carries its own RNA template [127]. Telomere length is very tightly regulated, with each organism and tissue having a characteristic range that is kept by tight homeostatic mechanisms [128]. The activity of telomerase must be coordinated with that of the regular replication machinery, in order to ensure proper replication of all the geneome. A genetic screen for deletion mutants that affect telomere length in S. cerevisiae identified Elg1 as a protein that participates in telomere length maintenance [44]. Deletion of ELG1 increases telomere length unraveling a role for Elg1 in the negative regulation of telomere elongation. This telomeric phenotype is not shared with the other alternative RLCs; Dctf18 mutants have short telomeres while Drad24 mutants exhibit no telomere length

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phenotype. This demonstrates that Elg1’s function in telomere length maintenance is distinct from the other RLCs. Elongation of telomeres can occur either by a telomerasedependent pathway or by an alternative pathway involving recombination between the repeated telomeric sequences. Deletion of ELG1 results in hyper-recombination (as discussed above), raising the possibility that the observed telomere elongation in Delg1 strains is a consequence of recombination. However this is not the case, as deletion of RAD52, a protein essential for homologous recombination, does not affect Delg1 mutant’s telomere length. On the other hand, deletion of EST2, the catalytic subunit of telomerase, prevents telomere elongation in the double mutant Delg1 Dest2. These results suggest that telomere elongation in the absence of Elg1 is due to telomerase activity [41]. The increase in telomere length by Delg1 also requires the DNA replication checkpoint [38]. Interestingly, Delg1 mutants, in addition of their long telomere phenotype, also display increased telomere position effect (TPE; also called telomere silencing). This last phenotype, however, seems to be independent of actual telomere length: Delg1 strains carrying a plasmid bearing the ELG1 gene have normal TPE and telomere length; if the complementing plasmid is lost, however, the increased silencing phenotype of Delg1 can be immediately be seen, long before telomeres are substantially elongated [41]. The mechanism that underlies telomere elongation in the absence of Elg1 still needs to be determined. Combined mutations of proteins that negatively regulate telomerase recruitment or activity such as Rif1 and Pif1 with Delg1 result in a synergistic increase in telomere length [41]. This suggests that Elg1 does not regulate telomerase directly via these pathways. Indeed, preliminary data from our lab suggests that the lack of PCNA-clamp unloading is the reason why Delg1 mutant strains display long telomeres (Fig. 2B). A mutant in which the two lysine residues of PCNA that undergo modifications are altered (pol30 K127R, K164R) fully suppresses the Delg1 telomeric phenotype (data not shown). Abolishing ubiquitination of PCNA by either mutating residue 164 alone (the only residue that can undergo ubiquitination) or by deleting RAD18 does not influence Delg1 telomeric length. These results together with the fact that deletion of ELG1 results in accumulation of chromatin bound PCNA (discussed above) suggest that accumulation of SUMOylated PCNA leads to the elongation of telomeres in these circumstances. The activity of telomerase must be coordinated with that of the DNA polymerases that replicate the genome; whereas telomerase elongates the leading strand (Gstrand), using its RNA molecule as a template, it is necessary to carry out lagging strand synthesis in order to complete the replication. This is accomplished by contacts between subunits of telomerase and the CST (Cdc13-Stn1-Ten1) complex, bound to the ssDNA overhang, which promote recruitment of Polymerase alpha [129]. We can postulate two main mechanisms that might explain the elongated telomeres in Delg1 mutants (Fig. 3): (1) failure to unload PCNA, and specifically SUMOylated PCNA, might prevent telomerase from stopping the synthesis of telomere repeats. Perhaps the unloading of PCNA serves as signal for completion of replication, and for exchange between telomerase and DNA polymerase that should in turn synthesize the C-strand. Alternatively, unloading of PCNA may be required for the loading of DNA polymerase bound to a new PCNA which might result in telomerase eviction. (2) A different mechanism by which lack of Elg1 may lead to elongated telomeres could be that spontaneous lesions created in Delg1 strains elicit a DDR response that, among other processes, leads to telomere elongation. Consistent with this view, mutations in replication checkpoint genes, such as DUN1 and DPB11 prevented telomere elongation in Delg1 strains [38]. Whereas one could argue that in the first scenario a normal checkpoint activation is necessary for full telomere extension by

A

Failure to unload PCNA Pol

Elg1 CST

B

Telomerase

Acvaon of DNA damage response

Elg1 DNA lesion?

ssDNA gap?

Telomerase

DDR

Fig. 3. Two alternative models for the role of Elg1 at telomeres. (A) Unloading of SUMOylated PCNA is a signal that regulates telomerase activity; in its absence telomeres elongate. (B) In the absence of Elg1, incomplete DNA replication triggers the DNA damage response, and subsequently telomere elongation.

the presence of SUMOylated PCNA at telomeres, it is hard to reconcile the second hypothesis with the suppression of telomere length observed in pol30 K127R K164R mutants. We therefore favor the first hypothesis. Formal demonstration will require further insights on the mechanisms coordinating leading and lagging strand synthesis at telomeres. 4.5. Chromatin In the nucleus of living cells, DNA is wrapped around histone octamers, thus forming nucleosomes, which in turn are folded to higher order structures, which make up chromatin. Much of the genome in higher eukaryotes is found in a repressed state, refractory to gene expression, called heterochromatin [130]. Heterochromatin is marked in higher eukaryotes by the presence of deacetylated and methylated histones and repressive protein complexes that bind these histones [130]. Three regions in the yeast genome undergo active silencing: ribosomal DNA, silent mating type cassettes (HML and HMR), and telomeres [131]. Gene silencing in HML and HMR is maintained due to the activity of the SIR complex: mutation in any one of the SIR2/ 3/4 genes leads to derepression of the silent mating type regions [131]. Sir2 works as an NAD histone deacetylase, deacetylating the tails of histones H3 and H4 and allowing recruitment and spreading of Sir3 and Sir4 [132]. Near telomeres, Rap1p, the major telomere binding protein in budding yeast, recruits Sir4p [133]. As in mating type silencing, several redundant mechanisms exist to recruit the SIR silencing complex. The SIR complex can spread to distances of 3–5 kb from the chromosome termini and affect the transcription of genes adjacent to telomeres, in what is termed ‘‘telomere silencing’’, ‘‘telomere position variegation’’ or ‘‘telomere position effect’’ [134]. Finally, Sir2 also plays an important role regulating silencing and recombination in the ribosomal gene cluster. At the rDNA Sir2 interacts with Net1 and Cdc14 to form the RENT complex. Loss of Sir2 function leads to loss of rDNA silencing and increased recombination between rDNA repeats [135]. Sir2, the founding member of the sirtuin family, provides a link between cellular metabolism and aging by virtue of its NAD+ dependent histone deacetylase activity [136]. One of the most conspicuous phenotypes of elg1 mutants is hyper-silencing at telomeres [41]. Although elg1 strains have

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elongated telomeres, the hyper-silencing phenotype could be separated from the long telomere phenotype, demonstrating a different mechanism of action [41]. A screen for high-copy number suppressors of the hyper-silencing phenotype resulted in the isolation of a truncated SIR3 gene known to act as a dominant negative allele that causes de-silencing, as well as clones carrying the mating type gene. Overexpression of the mating type locus caused in turn overexpression of STE5, a MAPK scaffold, and HOG1, a stress-activated MAPK, which affected Sir2 recruitment, thus demonstrating an intimate link between MAPK signaling and heterochromatin formation in yeast [137]. A third locus isolated in this screen was ASF1, encoding a histone chaperone. During DNA replication it is necessary to duplicate the number of nucleosomes; nucleosomes are thus disassembled in front of the moving fork, and re-assembled, together with new histones, behind the fork. Asf1 plays a role in that process (Fig. 2C) [138]. Newly synthesized histones deposited during DNA replication are acetylated at their N termini and in the core region. Lysine K56 acetylation is present on newly synthesized histone H3, accumulates during S phase, and is then removed either prior to or during mitosis in a process regulated by the redundant yeast sirtuins, Hst3p and Hst4p [139]. In hst3 hst4 mutants, K56 acetylation is observed in virtually 100% of histones throughout the cell cycle [140]. The importance of K56 deacetylation is evident from the high level of genomic instability observed in hst3 hst4 cells [141]. These cells activate a DNA damage checkpoint response due to the presence of chronic DNA damage and depend on a functional DNA damage checkpoint for viability. The temperature sensitivity, increased levels of recombination, chromosome loss and sensitivity to DNA damaging agents can be suppressed by replacing lysine 56 of histone 3 by arginine [140], demonstrating that all phenotypes result from excess acetylation at this lysine residue. A screen for mutants that suppress the temperature sensitivity of hst3 hst4 cells identified mutations in the large subunits of the three RLCs (Elg1, Ctf18 and Rad24). Interestingly, overexpression of the RFC1 gene had the same effect. As the RFC and RLCs share the small subunits (Rfc2–5), these results suggested that the suppression is due to increased activity of RFC. These strains still carried acetylated histones, indicating that the suppression was due to the ability to survive in the presence of constitutive H3K56Ac, rather than to an inability to create it. Importantly, deletion of CTF4 had the same effect, and epistasis analysis indicated that Ctf4 participates, together with ASF1 [140] and RTT109 [142] in the H3K56Ac pathway. Taken together, these results suggest that RLC activity in the presence of acetylated histones is detrimental for the cells. As the fork progresses, nucleosomes are removed in front of the moving replication machinery and new, acetylated histones are added in its wake, to restore DNA packaging. Once the DNA is packed, the acetylation needs to be removed, and this event may serve as a signal for other activities at the lagging strand, such as ligation of the Okazaki fragments (Fig. 2C). As with acetylation of cohesin molecules, there probably is a coordination between the loading/ unloading activity of the passing fork and deacetylation activity. Elg1 and Ctf4 may carry out alternative activities at this stage, thus explaining their synthetic lethality [67]. Preliminary results show that Elg1 and Ctf18 affect H3K56-overexpressing cells differently, suggesting again separate, although interconnected roles for the two RLCs. 5. Are all the phenotypes of Delg1 dependent on PCNA modification? While there is a very clear connection between Elg1, PCNA and SUMOylation, many of the results obtained point to phenotypes of Delg1 that do not depend on PCNA modifications. For example, the

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Brown lab showed that part of the phenotypes can be suppressed by expressing truncated Elg1 proteins that lack the N-terminal motif, necessary for Elg1-PCNA interactions [45]. Whereas it can be admittedly suggested that the Elg1-RLC may still be able to interact with SUMOylated PCNA through links provided by the small RFC subunits or other regions of the Elg1 protein, it is clear from genetic experiments that deleting ELG1 increases the sensitivity to DNA damaging agents of pol30-K127R, K164R mutants (which cannot be modified) [62]. This synergistic effect suggests that Elg1 may have roles that are independent of PCNA modifications. Consistently, the physical interaction of Elg1 with PCNA takes place even when PCNA cannot be modified [56]. These PCNA-modification-independent activities of Elg1 are presently the subject of intensive research. 6. Elg1 in mammals Orthologs of Elg1 were identified in many organisms, including humans. Human ELG1/ATAD5 has been shown to play an important role in maintaining genome stability in S phase [95,143]. Targeted gene knockdown of hELG1 results in spontaneous foci formation of g-H2AX, 53BP1 and phosphorylated ATM, all markers of spontaneous chromosomal breaks. hELG1 knockdown also leads to increased levels of recombination and chromosomal aberrations such as chromosomal fusions and inversions [95,143]. hELG1 was also found to link DNA replication stalling with apoptosis [144]. USP1 (ubiquitin-specific protease 1) was identified as a deubiquitinating enzyme for PCNA after DNA damage bypass [145]. The USP1/UAF1 complex reduces the accumulation of monoubiquitinated PCNA during normal cell division, which prevents mutagenesis by error-prone TLS polymerases. In response to damage that stalls DNA replication, USP1 is degraded, and consequently mono-ubiquitinated PCNA accumulates [146]. UAF1 is also a component of the Fanconi Anemia pathway in charge of deubiquitinating FANCD2 [94]. hELG1 was found in association with the USP1/UAF1 complex and down regulates PCNA mono-ubiquitination. Interestingly, hELG1 knockdown resulted in an increase in the level of PCNA mono-ubiquitination without apparently affecting the level of FANCD2 ubiquitination. Knockdown of the other RLCs did not have the same effect, again implying specific functions for Elg1 not shared with the other RLCs [95]. To investigate the role that Elg1 plays in the development of cancer, Myung and co-workers [147] attempted to knockdown the gene in mice. Homozygous Elg1m/m mice die as embryos, underscoring the important role played by this gene. Heterozygous mice can be created, but display a haploinsufficient phenotype: primary Elg1+/m fibroblasts show high levels of aneuploidy and other visible genome damage in culture. Elg1+/m mouse embryonic fibroblasts exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Moreover, 90% of heterozygous Elg1+/m mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, and these exhibited high levels of genomic alterations [147]. A search in human cancer databases identified somatic mutations of ELG1 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ELG1 function. These results suggest that the genome stability seen in the absence of proper ELG1 function can promote the development of cancer [147]. The human ELG1 gene is located on chromosome 17q11.2 next to the NF1 gene, defective in Neurofibromatosis type 1 patients. Neurofibromatosis is an autosomal dominant tumor promoting syndrome, whose hallmark symptom is multiple benign dermal neurofibromas. This syndrome exhibits inter- and intrafamilial variability, especially in its cancer incidence.

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Lately, several studies have found a higher cancer incidence in patients who have a micro-deletion that includes ELG1 [148]. These are approximately 10% of the neurofibromatosis patients. Taken together, these studies suggest that ELG1 is a tumor suppressing gene, and in its absence genome instability leads to tumorigenesis. It is possible that the high level of spontaneous genomic damage might be due to defects in the removal of ubiquitin from PCNA by ELG1–USP1–UAF1 after DNA damage bypass [19]. In this model the persistent presence of ubiquitinated PCNA on DNA could lead to fork collapse and abnormal recombination. Consistent with this possibility, higher levels of PCNA ubiquitination and Rad51 foci were observed in response to MMS in ELG1+/m MEFs compared to wild type MEFs. However, it is worth noting that recent studies have shown that the Usp1 knock-out mice, and the PCNA-K164R knock-in mice both have milder phenotypes compared to ELG1+/m mice. Therefore, some of the phenotypes of ELG1+/m might be due to additional roles of ELG1 (e.g. in SCC or Okazaki fragment maturation). Recently, it was found that mammalian PCNA undergoes SUMOylation [149,150], as well as modification by ISG15, another ubiquitin-like small molecule that is believed to serve in terminating PRR [80]. The interaction of hELG1 with these forms of modified PCNA has yet to be studied. 7. Summary We have shown that the Elg1-RLC plays important roles in a variety of processes that impinge on the integrity and stability of the genome (Fig. 2). Many of the phenotypes observed in Delg1 mutants seem to be related to its roles as a PCNA (and particularly SUMOylated PCNA) unloader [61]. However, Elg1 seems to have additional functions, which appear independent of PCNA modification. For example, a double mutant pol30-K127R, K164R Delg1 strains show higher sensitivity to DNA damaging agents than the single Delg1 or pol30-K127or pol30-K164 mutants [67]. This implies that each of the genes have additional functions. In addition, some of the Delg1 phenotypes (such as the suppression of temperature-sensitivity of hst3 hst4 mutants) cannot be suppressed by eliminating modifications in PCNA. At this stage, it is also unclear whether all the activities of the Elg1-RLC are mediated by SUMO and the Slx5/8 STUbL. It is also clear that many of the phenotypes of Delg1 strains (telomere length, gross chromosomal rearrangements and suppression of extra H3K56 acetylation) are abolished when the DDR, particularly the replicative branch of this checkpoint response, is inactivated. This has been interpreted many times to signify that the phenotypes are the result of eliciting the checkpoint (e.g. [36,38,45,151,152]). However, as we are discussing mechanisms, such as DNA replication, chromatin remodeling and sister chromatid cohesion, that are very central to the cell’s viability, an alternative explanation is that the DNA damage response is needed for viability under those dire circumstances. How conserved is the role of Elg1 during evolution? PCNA posttranslational modifications are common among yeast and mammalians, and seem to function in the regulation of DNA replication and repair in all organisms. Although the yeast Elg1 protein was found to preferentially interact with SUMOylated PCNA [56,63], its human counterpart was found to regulate the levels of PCNA ubiquitination, not SUMOylation [95]. However, it has to be noted that SUMOylated proteins are conspicuously hard to detect biochemically. hELG1’s role in PCNA de-ubiquitination is mediated by its interaction with the USP1/UAF1 complex, which has been shown to work in de-ubiquitination [95]. Interestingly, UAF1 contains a tandem repeat of SUMO-like domains at its Cterminus which bind to a SIM (SUMO-Interacting Motif) on hELG1 allowing the recruitment of the USP1/UAF1 complex to the ubiquitinated PCNA substrate. Thus, recruitment to PCNA may

be carried out by SUMO in yeast or SUMO-like proteins in humans, to carry out similar functions. RNF4, the human ortholog of the Slx5/8 STUbL, can be found at the replication fork, as well as at DNA damage sites, and participates in the degradation of many chromatin-associated factors [153]. However, a direct association with hELG1, as observed in yeast cells, has still to be established. Interestingly, human PCNA was found to interact with ESCO2, the human Eco1 homologue [106], and with hELG1 [95], suggesting that the function of Elg1 in sister chromatid cohesion may be conserved. In yeast deletion of ELG1 partially rescues the phenotypes of a temperature-sensitive eco1 mutant whereas its overexpression enhances its phenotypes [66]. It will be interesting to test in a mammalian system whether hELG1 overexpression can also suppress cohesion defects caused by lack of ESCO2. Conflict of interest statement The authors report no conflicts of interest to disclose. Acknowledgements We thank members of the Kupiec lab for their suggestions, commentaries and support. Research on Elg1 in the Kupiec lab is supported by a grant from the Israel Science Foundation. References [1] J. Lee, Y. Lee, M.J. Lee, E. Park, S.H. Kang, C.H. Chung, K.H. Lee, K. Kim, Dual modification of BMAL1 by SUMO2/3 and ubiquitin promotes circadian activation of the CLOCK/BMAL1 complex, Mol. Cell. Biol. 28 (2008) 6056–6065. [2] K.A. Nyberg, R.J. Michelson, C.W. Putnam, T.A. Weinert, Toward maintaining the genome: DNA damage and replication checkpoints, Annu. Rev. Genet. 36 (2002) 617–656. [3] G.L. Moldovan, B. Pfander, S. Jentsch, PCNA, the maestro of the replication fork, Cell 129 (2007) 665–679. [4] C. Hoege, B. Pfander, G.L. Moldovan, G. Pyrowolakis, S. Jentsch, RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO, Nature 419 (2002) 135–141. [5] H.D. Ulrich, S. Jentsch, Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair, EMBO J. 19 (2000) 3388–3397. [6] D. Branzei, F. Vanoli, M. Foiani, SUMOylation regulates Rad18-mediated template switch, Nature 456 (2008) 915–920. [7] H. Windecker, H.D. Ulrich, Architecture and assembly of poly-SUMO chains on PCNA in Saccharomyces cerevisiae, J. Mol. Biol. 376 (2008) 221–231. [8] E. Papouli, S. Chen, A.A. Davies, D. Huttner, L. Krejci, P. Sung, H.D. Ulrich, Crosstalk between SUMO and ubiquitin on PCNA is mediated by recruitment of the helicase Srs2p, Mol. Cell 19 (2005) 123–133. [9] B. Pfander, G.L. Moldovan, M. Sacher, C. Hoege, S. Jentsch, SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase, Nature 436 (2005) 428–433. [10] P. Stelter, H.D. Ulrich, Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation, Nature 425 (2003) 188–191. [11] A. Halas, A. Podlaska, J. Derkacz, J. McIntyre, A. Skoneczna, E. SledziewskaGojska, The roles of PCNA SUMOylation, Mms2-Ubc13 and Rad5 in translesion DNA synthesis in Saccharomyces cerevisiae, Mol. Microbiol. 80 (2011) 786–797. [12] J. Majka, P.M. Burgers, The PCNA-RFC families of DNA clamps and clamp loaders, Prog. Nucleic Acid Res. Mol. Biol. 78 (2004) 227–260. [13] C.M. Green, H. Erdjument-Bromage, P. Tempst, N.F. Lowndes, A novel Rad24 checkpoint protein complex closely related to replication factor C, Curr. Biol. 10 (2000) 39–42. [14] L.A. Lindsey-Boltz, V.P. Bermudez, J. Hurwitz, A. Sancar, Purification and characterization of human DNA damage checkpoint Rad complexes, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 11236–11241. [15] J. Majka, S.K. Binz, M.S. Wold, P.M. Burgers, Replication protein A directs loading of the DNA damage checkpoint clamp to 50 -DNA junctions, J. Biol. Chem. 281 (2006) 27855–27861. [16] J. Majka, P.M. Burgers, Yeast Rad17/Mec3/Ddc1: a sliding clamp for the DNA damage checkpoint, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 2249–2254. [17] G.O. Bylund, J. Majka, P.M. Burgers, Overproduction and purification of RFCrelated clamp loaders and PCNA-related clamps from Saccharomyces cerevisiae, Methods Enzymol. 409 (2006) 1–11. [18] C.Y. Bonilla, J.A. Melo, D.P. Toczyski, Colocalization of sensors is sufficient to activate the DNA damage checkpoint in the absence of damage, Mol. Cell 30 (2008) 267–276. [19] G.I. Karras, M. Fumasoni, G. Sienski, F. Vanoli, D. Branzei, S. Jentsch, Noncanonical role of the 9-1-1 clamp in the error-free DNA damage tolerance pathway, Mol. Cell 49 (2013) 536–546.

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